THE JOURNAL OF BIOL.OGICAL. CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

RNA Editing of Apolipoprotein B mRNA

Vol. 265, No. 12. Issue of April 25, pp. 6811-6816, 1990 Printed in U.S. A.

SEQUENCE SPECIFICITY DETERMINED BY IN VITRO COUPLED TRANSCRIPTION EDITING*

(Received for publication, December 14, 1989)

San-Hwan ChenS, Xiaoxia Li$, Warren S. L. LiaoQ, June H. Wu$, and Lawrence ChanS From the SDepartments of Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030 and the SDepartment of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Apolipoprotein (apo) B-48 mRNA is produced by in viva RNA editing which involves a C+U conversion of the first base of the codon CAA for Gln-2153, changing it to UAA, an in-frame stop codon. We have reproduced the editing reaction in vitro using nuclear extracts. Efficient RNA editing was demonstrated by using apoB mRNA segments as substrate or in a coupled transcrip- tion-editing reaction using apoB minigenes as tem- plate. ApoB minigenes were constructed by ligating the adenovirus major late promoter to a fragment of apoB- 100 DNA containing the editing site and used for the transcription-editing reaction. We defined the se- quence specificity of the editing reaction using site- specific single and multiple base mutants constructed by the polymerase chain reaction. Among 22 different mutant apoB- 100 minigene constructs containing mu- tations in the bases immediately flanking the edited C- 6666, 20 were edited in the coupled transcription- editing reaction. The results suggest a relatively lax sequence specificity for apoB mRNA editing. Our ob- servation may have important implications for apoB- 48 biogenesis as well as for the editing process as a general biologic regulatory mechanism.

RNA editing is a molecular biological phenomenon whereby the primary structure of an RNA transcript is altered by mechanisms other than splicing (l-4). The first description of this process involved the addition of nongenomically en- coded uridine (U) residues to mitochondrial mRNAs in the kinetoplastid protozoa (5). Subsequently, U residues have been found to be removed from some transcripts (6, 7). Recently, Thomas et al. (8) described the addition of two nontemplated G residues in Paramyxovirus SV5 which joined two open reading frames to produce the P protein. An analo- gous situation was also described in measles virus where variable numbers of Gs were inserted into the molecule (9). The only putative RNA editing described in mammals in- volves the conversion of a C to a U residue in apoliprotein (apo) B mRNA in the small intestine (10, 11); editing was also demonstrated recently in cells transfected with apoB gene constructs (12, 13).

ApoB is a major protein constituent of plasma lipoproteins. The plasma concentration of apoB shows a strong direct correlation with the development of coronary artery disease (14, 15). Plasma apoB exists in two major forms (16), apoB-

* This work was supported by National Institutes of Health Grants HL 27341 (to L. C.) and AR 38858 (to W. S. L. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

100 and apoB-48, which contain 4536 and 2152 amino acid residues, respectively (10, 11, 17-21). In humans, apoB-100 is the product of a 14-kb’ mRNA in the liver. ApoB-48 is the product of an intestinal mRNA of adult humans which is identical in structure to apoB-100 mRNA except for a single C+U base substitution involving C-6666, the first base of the codon CAA for Gln-2153, changing it to UAA, a stop codon (10, 11).

The mechanism behind the C-U conversion is unknown. In this study, we have reproduced the RNA editing in vitro using a nuclear extract and a template consisting of either a segment of apoB DNA (i.e. coupled transcription editing) or apoB mRNA (i.e. direct RNA editing). By altering the nu- cleotide sequence of the template by site-specific mutagenesis, we have defined the sequence specificity of the editing reac- tion. The sequence specificity of the reaction was found to be relatively lax, which has important implications for the po- tential role of RNA editing in the regulation of gene expres- sion.

MATERIALS AND METHODS

Primer Extension Assay for RNA Editing-Primer extension in the presence of dideoxy GTP was used as a rapid assay for the absence (i.e. unedited template) or presence (i.e. edited template) of primer- extended product extending beyond C-6666. An oligonucleotide with the sequence 5’-AATCATGTAAATCATAACTATCTTTAATA- TACTG-3’ (34-mer) was used as primer. Primer extension was per- formed essentially as described by Driscoll et al. (22). The primer extension products were separated by electrophoresis on a 12% poly- acrylamide sequencing gel and exposed to Kodak XAR-5 x-ray film for varying periods of time.

Rut Liver Nuclear Extract Preparation-All manipulations were performed in the cold, and all solutions, tubes, and centrifuges were chilled to 0 “C. Rat liver nuclear extracts were prepared essentially as described by Gorski et al. (23). We normally obtained approxi- mately lo-15 mg of nuclear protein/l5 g of adult rat liver. Nuclear extracts from HeLa and Hep3B cells were prepared exactly as de- scribed by Shapiro et al. (24).

Coupled in Vitro Transcription-editing Reactions-An apoB-100 minigene was constructed by ligating the adenovirus major late pro- moter to the 5’ end of an apoB-100 DNA segment corresponding to different lengths of apoB mRNA identified in the legend to Table I (Fig. 2A ). In uitro transcription reactions (50 ~1) contained 3.0 pg of circular DNA template and 3-5 mg/ml nuclear protein extract in a buffer containing 25 mM Hepes (pH 7.6), 50 mM KCl, 6 mM MgCl,, 0.6 mM each of ATP, CTP, GTP, and UTP, 12% glycerol, and 1 ~1 of RNasin (40 units, Promega Biotech). After 1 h of incubation at 30 “C, the reactions were terminated by the addition of 380 ~1 of stop buffer (50 mM Tris-HCl, pH 7.5, 1% sodium dodecyl sulfate, and 5 mM EDTA). Ten pg of Proteinase K was then added, and the reaction was incubated at 65 “C for 30 min. After Proteinase K treatment, the RNA was extracted twice with equal volumes of phenol and chloro- form and precipitated by ethanol with 10 mg/ml tRNA carrier.

’ The abbreviations used are: kb, kilobase( Hepes, 4-(2-hydrox- yethyl)-l-piperazineethanesulfonic acid; PCR, polymerase chain re- action.

6811

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6812 Coupled Transcription Editing of ApoB mRNA

Quantitation of H-G/n and B-Stop Sequences-Nucleic acids from the reaction mixture were treated with Sau3A1, and the enzyme was removed by Proteinase K treatment. The sample was phenol-ex- tracted and the nucleic acids precipitated in ethanol. They were resusoended in CsCl (0.4 z/ml in 10 mM Tris. DH 7.5. 1 mM EDTA). layered on top of a CsCl cushion (5.7 M CsCl in 0.1 M EDTA, pH 7.5) and centrifuged at 35,000 rpm in an SW 50 rotor at 20 “C for 16 h. The RNA pellet was recovered and treated with RQ DNase I (Pro- mega Biotech) (6 units/3-pg sample) in 40 mM Tris, pH 8, 10 mM NaCI, 6 mM MgCln at 37 “C for 30 min. The reaction was stopped by incubation for 30 min at 37 “C with Proteinase K (200 pg/ml) and adiustment of the buffer to 10 mM Tris. DH 8.0, 5 mM EDTA. and 0.5% sodium dodecyl sulfate. The RNA’ was then extracted’with phenol/chloroform and precipitated in ethanol (25).

1’ 1 2 3 4 5 6 4’ 5’6’ “”

PCR Cloning-Hybridization Assay-RNA samples were amplified bv the nolvmerase chain reaction (26) (PCR). fractionated on 2% agarose’geis, and the amplified products cut out from the gels. They were then cloned into the EcoRI/BamHI site of pGEM-3Z in Esche- richia co/i host JM109. Colony hybridization on nitrocellulose filters to B-Gln or B-Stop oligonucleotides was performed at 50 “C and the filters exposed to x-ray films XAR-5 for 6-20 h. The sequences of the oligonucleotides used for priming the PCR reaction and for hybridi- zation are: for the human RNA in Table I. PCR primers 5’- GGAATTCTCACAAAAAAGTATAGAA-3’ and 5’-CGGATCCA- CTTTTGTTAAAATCAA-3’; the hybridization probes were B-Stop orobe. 5’-TACTGATCAAATTATATCA-3’ and B-Gln urobe. 5’- TACTGATCAAATTGTATCA-3’. In each experiment, randomly se- lected colonies that hybridized to the B-Gln- or B-Stop-specific oligonucleotides were directly sequenced. They all showed 100% concordance with the hybridization data. DNA sequencing was per- formed directly on double-stranded DNA by the method of Sanger et al. (27).

Site-specific Mutagenesis of ApoB-100 Sequences--B-loo260 cDNA inserts were used as templates for site-specific mutagenesis (Fig. 2B). These inserts had a unique BclI site four bases downstream from C- 6666. The PuuII/BclI insert was amplified by PCR. The 5’-oligonu- cleotide primer overlapped the PuuII site. The 3’-primer which over- lapped both the BclI site and C-6666 and neighboring bases was the “mutagenic” primer that included the mutated bases. The PCR prod- uct was treated with PvuII and BclI, ligated to the wild type BclI/ XbaI fragment. and subcloned into the PuuIIIXbaI site of the AdB- 100 plasmid (25). The sequences of all the mutant clones were confirmed by direct nucleotide sequencing (27).

RESULTS AND DISCUSSION

Editing of ApoB RNA in Vitro-In our initial testing of RNA editing in uitro, we incubated T7 transcripts of cloned apoB-100 cDNA segments containing C-6666 with a nuclear extract from rat liver, a tissue known to contain large amounts of edited apoB mRNA (28, 29). The RNA was then purified and analyzed by two different assays: (i) the PCR cloning- hybridization method described under “Materials and Meth- ods” and (ii) a primer extension assay similar to the one described recently by Driscoll et al. (22). The primer extension method is rapid and moderately sensitive, detecting down to 1% of C+U conversion in standard RNA mixtures of 0.1 pg. Unfortunately it is not linear in the lower concentration range, and its sensitivity is also limited. The PCR cloning- hybridization procedure was much more sensitive. For exam- ple, while editing was demonstrated with 100 and 10 ng of apoB RNA transcripts in the reaction, the primer extension failed to detect the editing of an in vitro apoB transcript when only 1 ng was used in the reaction (Fig. 1). In contrast, when the PCR cloning-hybridization assay was used, editing was detected with all three concentrations of input RNA (Table I, A). It was more efficient in the reaction using 10 ng than those using higher (100 ng) or lower (1 ng) amounts of input RNA. The latter two RNA concentrations were edited to about the same extent. The fact that editing was detected in the lOO- and lo-ng but not in the l-ng reaction in the primer extension assay indicates that the B-Stop signal was a func- tion of the total amount of B-Stop sequences generated, which

tsil - c r-54-mer

44 -43-mer

w mom0 --00700 -’ -’ 0 v-0 -0

Iv1 v-

B-48 B-1 00 FIG. 1. Primer extension assay on apoB-Gin and apoB-Stop

RNAs incubated with rat liver nuclear extracts. A 34-base oligonucleotide primer (apoB 6708-6675) was 5’ end-labeled with “P by polynucleotide kinase and [Y-~P]ATP and hybridized to the apoB mRNA. Primer extension was performed in the presence of high concentrations of dideoxy GTP such that it terminated at the next C. Termination at C-6666 would produce a 43-mer and at C-6655 would produce a 54-mer. Lanes I-3 and 4-6 represent different amounts of apoB-48 (B-Stop) and apoB-100 (B-Gln) RNA in the in vitro editing reaction, respectively. These lanes were exposed to a Kodak XAR-5 x-ray film for 16 h. Lanes I’ and 4’-6’ were repeat exposures of the corresponding numbered lanes for 48 h in the presence of two intensifying screens. A 54-mer (B-Stop) band is readily identified in the 0.3 ng B-48 lone (I ‘); the lowest concentration of B-100 RNA that produces an identifiable band of the 54-mer in the B-100 RNA reaction is 10 ng (lane 5’).

is in turn dependent on the total input RNA in the assay. Therefore, it is not an accurate assay for the extent of editing, and its sensitivity is much inferior to the PCR cloning- hybridization assay, which is independent of the input RNA concentration. Thus, the latter assay alone was used in all subsequent experiments.

Both nuclear extract and apoB mRNA were needed for the reaction. Incubation of apoB mRNA with buffer alone or with heat-inactivated nuclear extract failed to show any C+U conversion. The authenticity of the edited product was con- firmed by direct sequencing. Of all the Cs in the B-mRNA,kt,, only one (C-6666 of the codon CAA for Gln-2153) was con- verted to a U; none of the other Cs were changed. Also, this base substitution is the only one observed; by direct sequenc- ing, there is no other type of base changes in the edited RNA.

Coupled Transcription Editing of ApoB-100 Minigene-The nuclear extract (23) used for RNA editing had been found earlier to be active in transcribing a number of mammalian genes (including the albumin and serum amyloid A genes, data not shown). We tested its ability to direct the coupled transcription editing of an apoB-100 minigene.

An apoB-100 minigene was constructed by ligating the adenovirus major late promoter to the 5’ end of an apoB-100 DNA segment corresponding to different lengths of apoB mRNA (Fig. 2A). The AdB-100 minigene was incubated with the nuclear extract. The RNA product was purified and as- sayed for the relative proportions of B-Stop versus B-Gln transcripts by the PCR cloning-hybridization assay. Authen- ticity of the products was further confirmed by direct sequenc- ing. It is evident that the nuclear extract can transcribe the AdB-100 minigene constructs as well as edit the transcripts under these conditions (Table I, B). The nuclear extract was somewhat more effective in the coupled transcription-RNA editing of AdR-100260 than the two longer constructs. Fur- thermore, both the supercoiled and linearized minigenes were transcribed and edited although the efficiency may be slightly higher with the supercoiled substrate. As in the case of RNA substrates, the only base change detected in these coupled transcription-RNA editing reactions was C-6666.

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Coupled Transcription Editing of ApoB mRNA 6813

TABLE I

Direct RNA editing and coupled transcription editing in vitro

Reaction conditions” % editing ((B-Stop/(B-Stop + B-Gin)) x 100)

A. Direct RNA editing in uitro Extract + B-mRNAm,

100 ng of mRNA 0.76 (10/1320)b 10 ng of mRNA 1.71 (52/3037)

1 ng of mRNA 0.94 (15/1595) Buffer + 10 ng of B-mRNA1kb 0 (0/>8000) Extract + buffer NP’

B. Coupled transcription-RNA editing in vitro Supercoiled AdB-100z6” + rat liver extract 1.47 (194/13394) Supercoiled AdB-lOOso + rat liver extract 0.52 (53/10253) Supercoiled AdB-100m, + rat liver extract 0.48 (49/10169) Linear AdB-100260 + rat liver extract 0.84 (69/8205) Linear AdB-lOOso + rat liver extract 0.28 (24/8680) Linear AdB-lOO,kb + rat liver extract 0.51 (48/9460) Supercoiled AdB-100z6” + buffer NP’ Buffer + rat liver extract Supercoiled AdB-lOOzGO + HeLa extract 0 (07:25) Supercoiled AdB-100260 + Hep3B extract 0 (O/3431)

“AdB-100260 is the construct shown in Fig. 2A. It contains bases 6552-6815 of apoB-100 mRNA. AdB-lOOso contains bases 655227034, and AdB-lOOrkb, 6552-7573. B-mRNAm, is the in vitro T7 transcript from AdB-lOOm,. For direct RNA editing, a T7 transcript of linearized AdB-100rkb was used as substrate. For coupled transcription editing, either supercoiled or linear (i.e. cut by XbaI) DNA constructs were directly used as substrates.

bActual number of clones hybridizing to B-Stop- and B-Gln-specific oligonucleotide probes are given in parentheses: B-Stop/(B-Stop + B-Gin).

’ NP, no PCR product detected. In the coupled transcription-editing experiments, the treatment of the reaction mixture described under “Materials and Methods” completely removed any AdB-100 DNA that could be amplified by the PCR.

P”l II BLI I xd2. I

FIG. 2. AdB-100zeo DNA constructs for coupled transcrip- tion editing. A, wild type AdB-100260 construction. The adenovirus (Ad) 2 major late promoter was ligated to a segment of apoB-100 cDNA (bases 6552-6815). The hybrid adenovirus promoter apoB- 1OO260 is inserted into the EcoRI/XbaI site of pGEM-3Z. The regions of the apoB mRNA included in AdB-10026a, AdB-lOOsoo, and AdB- 100m,are specified in the legend in Table I. B, site-specific mutagen- esis of apoB-100 sequences in the construction of AdB-10026,1 mutants. The mutant base is indicated by an open circle. The position of C- 6666 is indicated by a C.

In both the direct RNA-editing and the coupled transcrip- tion-editing experiments, misincorporation in the PCR as the cause of the C-&J substitution was excluded by the following: (i) in the direct editing experiments, incubation of the RNA in buffer alone did not produce any B-Stop sequences assayed by the PCR cloning-hybridization technique; furthermore, primer extension assay of the RNA products without PCR amplification confirmed the editing reaction; (ii) in the cou- pled transcription-editing experiments, the PCR cloning-hy- bridization assay performed directly on the DNA template (i.e. AdB-1002& showed 100% B-Gln sequences; and (iii) direct sequencing of multiple cloned B-Gln or B-Stop PCR products revealed no other base substitutions.

Comparison of the direct RNA-editing and coupled tran- scription-editing experiments indicates that the two reactions were comparable in efficiency. The fact that the editing re- action occurred under cell-free conditions using nuclear ex- tracts suggests that RNA editing in uiuo may occur in a similar manner, i.e. as a coupled reaction in the nucleus. Therefore, we used the coupled transcription editing of supercoiled AdB- 100 minigenes to analyze the tissue and sequence specificity of the reaction.

In mammals, there is species- and tissue-specific variation in the efficiency of RNA editing. We tested the ability of nuclear extracts from three different tissues to perform the coupled transcription-RNA editing (Table I, B). All three extracts efficiently transcribed the apoB minigene. However, only nuclear extracts from rat liver edited the transcript. The activity of the extracts from HeLa or Hep3B cells was unde- tectable. This observation correlates with the presence of substantial amounts of apoB-48 mRNA in rat liver RNA and its absence in Hep3B RNA (data not shown); HeLa cells normally do not produce any apoB mRNA.

Sequence Specificity of ApoB mRNA Editing in Vitro-The sequence specificity of the coupled transcription-editing re- action was examined by site-directed mutagenesis of the AdB- 1OO26o DNA minigene (Fig. 2B). We constructed 22 different

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6814 Coupled Transcription Editing of ApoB mRNA

mutant apoB-100 minigenes, including constructs that con- tain single or multiple base substitutions in the bases imme- diately flanking C-6666, as well as one construct that contains a single-base insertion, and another, a single base deletion. The efficiency of editing was quantified by the PCR cloning- hybridization assay and compared with the editing efficiency of wild type Ad13-100Z60. The most obvious conclusion that can be drawn from these in uitro mutagenesis experiments is that the reaction is promiscuous with respect to the sequence of the RNA substrate; of the ‘22 mutant RNA sequences, all except two were edited by the nuclear extract in vitro (Table II). The results obtained in Table II were all confirmed by direct sequence analysis (data not shown).

In mutants u-n, the three bases flanking the 5’ and 3’ sides of C-6666 were mutated individually, producing transi- tion mutants (a+) or transversion mutants (g+n). These

constructs were edited in vitro with varying efficiency. For transition mutants, enhanced editing efficiency was observed for the mutant containing CGA instead of CAA (mutant a, which had a a-fold increase in efficiency); mutant T-6669-C (mutant c) was also edited somewhat more efficiently (-1.5- fold) than wild type. Mutant A-6665+G (mutant d) was edited with normal efficiency. The two other constructs con- taining transition mutations (mutants e and f) were edited with an efficiency of 50% of wild type. Three of the transver- sion mutants, h, g, and k, were edited with markedly, moder- ately, and slightly reduced efficiency, respectively. The other transversion mutants were edited with the same (mutants i, j, and 1) or higher efficiency (mutants m and n) compared with wild type. As noted below, introduction of single Cs in the proximity of C-6666 appears to stimulate editing in some instances.

TABLE II RNA editing efficiency of ApoB-100 mutants

Coupled transcription editing was performed using adenovirus major late promoter-apoB-100, AdB-10026o. The mutant minigenes were identical to the wild type with base substitutions shown. Asterisks represent identical residues. C-6666 is marked by an arrow. Following incubation, RNA was purified and analyzed by the PCR cloning- hybridization method as described under “Materials and Methods.” Clones that hybridized to the B-Gln- or B- Stop-specific oligonucleotides were confirmed by direct DNA sequencing.

Mutant sequence Efficiency”

1 Wild type ATA CAA TTT ++++ Transition mutants

E

l ** . G . *.* ++++++

*** **G * t t ++

:

t** *** C** +++++

“G *** l ** ++++

r ”

*c* l ** *** +++

G.’ l ** **a +++

Transversion mutants g *t* ST* St* ++ h *** *a’,- Sat + i *** *ii A’* ++++

j *‘T 1.. *It ++++ k *A* **t l ** +++

1 T.. t . * l ** ++++

m **. *c* et* ++++, b + : b +++++=

Dotble mutants **t **c *** +++++ , b ++ b

Transition 0 **G .G. tea ++ P l ** ‘GG *** ++++++

Transversion 4 “T *T* et* 0 r *t* *cc *** ++++,b+,b+;b+,dO,dO,dOd

Deletion, s *** ‘[I’ **a 0 Insertion, t .**c*** *a* ++,b+,b+= Multiple mutants

Transitions, u GCG *** ccc ++ Mixed. v GCT *** ccc +++

a The estimated efficiencies of the mutant AdB-100 sequences are normalized to the wild type editing efficiency by the following formula: (% B-Stop in mutant/% B-Stop in wild type) X 100. Editing efficiencies are: O%, 0; <25%, +; Z-50%, ++; 50-75%, +++; 75-125%, ++++; 125-200%, +++++; >200%, ++++++. By definition, wild type is taken as 100% or ++++. The actual percent B-Stop in wild type AdB-100260 transcripts varies from 0.6 to 2% in individual experiments. Each mutant was assayed in three separate experiments with consistent results. For each mutant, two separate oligonucleotide probes, one for the unedited C and one for the edited T, were used for specific hybridization to each C residue examined. In individual experiments, the total number of PCR-generated apoB clones was usually 4,000-10,000 with a range of 2,000-20,000. In the experiments where no editing was detected (mutants q and s) a total of over 20,000 colonies in three separate experiments was found to hybridize only to B-Gln oligonucleotides but not to the B-Stop oligonucleotides; in the same experiments editing was demonstrated with other constructs. Furthermore, the same B-Stop oligonucleotide probes produced strong hybridization signals to colonies of constructs containing a C-666&T mutation generated by site-directed mutagenesis.

b The multiple results in these constructs refer to the different Cs in close proximity to, and including C-6666, that were edited, starting with the 5’-most C.

’ Simultaneous editing of the neighboring Cs. d Simultaneous editing of C-6666 + C-6667, C-6667 + C-6668, C-6666 + C-6668, and C-6666 + C-6667 + C-

6668, in that order.

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Coupled Transcription Editing of ApoB mRNA 6815

Four double mutants tested were found to be edited with drastically different efficiencies. One, mutant p, which had CGG substituted for CAA, had a 2-3-fold increase in editing efficiency compared with wild type. Another, mutant r, which had CCC substituted for CAA, was edited at normal efficiency. One double transversion mutant (q) containing the mutated bases A-6665-T and A-6667+T was consistently not edited in vitro in three separate experiments. This complete inhibi- tion of editing should be contrasted with the roughly 50% editing efficiency of the corresponding double transition mu- tant (0). Complete inhibition of editing was also observed for the single deletion mutant, s, where one of the two As follow- ing C-6666 was deleted. In the insertion mutant, t, where an extra C was inserted next to C-6666, both Cs were edited with reduced efficiency.

The fact that the vast majority of mutations do not seriously impair editing efficiency suggests that the sequence require- ment of this reaction is not very stringent. To test this hypothesis, we constructed two 6-base substitution mutants where the two codons flanking the CAA are mutated, being replaced completely by transitional substitutions (mutant u) or five transitional substitutions and a single transversional substitution (mutant v). Transcripts from both constructs were edited with an efficiency approximately one-third (for mutant u) and two-thirds (for mutant v), respectively, of that of wild type.

In a number of the mutants that we studied, one or more Cs were introduced in the sequence in close proximity to C- 6666. These constructs include mutants m, n, r, and t. Like C-6666, most of the neighboring Cs were also found to be edited in vitro. It is interesting that introduction of a single C immediately next to C-6666 stimulated editing when the relative position of C-6666 was not altered (mutant m). Both Cs in mutant m were simultaneously edited with high effi- ciency. In mutant t, where there was an insertion, there was moderate impairment of editing efficiency, the 3’ C being affected more than the 5’ C. In comparing the relative effi- ciency with which each C was edited in individual mutants, there appears to be some general pattern. In three instances where the relative position of C-6666 was not changed (mu- tants m, n, and r), the original C-6666 was edited much more efficiently than the Cs at the neighboring positions. Mutant r contains a substitution of the triplet CCC for CAA. In this case, the first C-6666 was found to be edited with normal efficiency, whereas the two 3’ Cs, C-6667 and C-6668, were edited poorly. Simultaneous editing of the two 5’ Cs was also observed, though infrequently. These results suggest that there may be a distance effect between the edited C and some flanking DNA sequence. Results with mutants m, n, and r suggest that a minimal distance between C-6666 and some 3’ sequence or a maximal distance between this base and some 5’ sequence is preferred for efficient editing. The slightly more efficient editing of the 5’ C of the insertion mutant t is difficult to interpret because there is significant impairment of editing of both Cs.

Implications of Sequence Specificity of ApoB mRNA Edit- ing-We have found that a rat liver nuclear extract not only can edit synthetic apoB RNA in vitro (Table I, A), as was demonstrated recently by Driscoll et al. (22), but also could perform coupled transcription editing of a minigene construct in which the in vitro nascent apoB transcript was edited with fidelity (Table I, B). RNA editing is likely a nuclear event in vivo. The system described here may mimic the in vivo situ- ation better than one that uses a synthetic RNA as substrate. We have tested synthetic RNAs corresponding to each of the mutant constructs in direct editing experiments. All of them,

including mutants q and s, were edited in vitro, which suggests that coupled transcription editing may have a more stringent sequence requirement than direct RNA editing.

The ability to introduce multiple mutations to apoB RNA without seriously reducing the editing efficiency indicates that specific base pairing or hydrogen bonding involving the bases immediately flanking C-6666 is not required for recognition by the putative cytidine deaminase enzyme. The relative laxity of the structural requirement is also supported by the fact that when additional C residues were introduced to the vicinity of C-6666, they were edited often to the same extent as C-6666. On the other hand, since mutants q and s, which contained double transversions and a single deletion, respec- tively, were consistently not edited in the coupled transcrip- tion editing reaction, there is definitely a preferred RNA structure recognized by the enzyme.

Although most of the mutant apoB RNA sequences were edited with normal or reduced efficiency, two of the constructs (mutants a and p) were consistently edited at an efficiency approximately 3-fold that of wild type. Synthetic transcripts of both mutants that were fed into the nuclear extracts were also edited efficiently (data not shown). It is possible that both apoB RNA mutants are deleterious to the organism. Mutant a, which contains CGA in place of CAA, would be edited so efficiently in the liver that insufficient apoB-100, a physiologically important protein, would be produced. In con- trast, editing of mutant p, which contains CGG instead of CAA, would produce an Arg+Trp substitution and not a stop codon, and no apoB-48 would be produced in the intestine. For these reasons, we speculate that both mutations, if they occurred, would be eliminated from the population.

Recently, an analogous RNA-editing mechanism involving C-U conversions has been described as a common phenom- enon in plant mitochondria (30, 31). In our current analysis, the relatively loose stringency of the editing reaction suggests that other RNAs may also be edited in vivo. Thus, in mammals as in plant mitochondria, RNA editing may not be a unique biologic phenomenon confined to apoB.

Acknowledgments-We thank Dr. Wen-Hsiung Li for discussion and critical readings of the manuscript, Hui-Jia Zhu for technical assistance, and Sally Tobola for expert secretarial assistance.

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REFERENCES

Simpson, L., and Shaw, J. (1989) Cell 57,355-366 Eisen, H. (1988) Cell 53, 331-332 Maizels, N., and Weiner, A. (1988) Nature 334, 469-470 Lamond, A. L. (1988) Trends Biochem. Sci. 13,283-284 Benne, R., Van Den Burg, J., Brakenhoff, J. P. M., Sloof, P., Van

Boom, J. H.. and Tromp. M. C. (1986) Cell 36.819-826 Shaw, J. M., Feagin, J. K:, Stuart, K.,‘and Simpson, L. (1988)

Cell 53,401-411 Feagin, J. E., Abraham, J. M., and Stuart, K. (1988) Cell 53,

412-422 Thomas, S. M., Lamb, R. A., and Paterson, R. G. (1988) Cell 54,

891-902 Cattaneo, R., Kaelin, K., Bacgko, K., and Billeter, M. A. (1989)

Cell 56,759-764 Powell, L. M., Wallis, S. C., Pease, R. J., Edwards, Y. H., Knott,

T. J., and Scott, J. (1987) Cell 50, 831-840 Chen, S.-H., Habib, G., Yang, C.-Y., Gu, Z.-W., Lee, B. R., Weng,

S.-A.. Silberman. S. R.. Cai. S.-J.. Deslvoere. J.. Rosseneu. M.. Gotto, A. M., Jr.; Li, W.-H.: and Chan,“L. (198i) Science i38; 363-366

Davies, M. S., Wallis, S. C., Driscoll, D. M., Wynne, J. K., Williams, G. W., Powell, L. M., and Scott, J. (1989) J. Biol. Chem. 264, 13395-13398

Bostrom, K.,.Lauer, S. J., Poksay, K. S., Garcia, Z., Taylor, J. M.. and Inneraritv. T. L. (1989) J. Biol. Chem. 264. 15701- 15?08

_

Brunzell, J. D., Sniderman, A. D., Albers, J. J., and Kwiterovich,

by guest on June 11, 2018http://w

ww

.jbc.org/D

ownloaded from

6816 Coupled Transcription Editing of ApoB mRNA

P. O., Jr. (1984) Arteriosclerosis 4, 79-83 15. Freedman, D. S., Srinivasan, S. R., Shear, C. L., Franklin, F. A.,

Weber, L. S., and Berenson, G. S. (1985) N. Engl. J. Med. 316, 721-726

16. Kane, J. P. (1983) Annu. Rev. Physiol. 45,637-650 17. Chen. S.-H.. Yane. C.-Y.. Chen. P.-F.. Setzer. 0.. Tanimura. M..

Li, ‘W.-H.; Got& A. Ml, Jr., and Chan, L. (i986) J. Biol. &em: 261,12918-12921

18. Knott, T. J., Pease, R. J., Powell, L. M., Wallis, S. C., Rall, S. C., Jr., Innerarity, T. L., Blackhart, B., Taylor, W. H., Marcel, Y., Milne, R., Johnson, D., Fuller, M., Lusis, A. J., McCarthy, B. J., Mahlev, R. W., Levy-Wilson. B.. and Scott, J. (1986) Nature 3i3, 734-738 -

19. Law, S. W., Grant, S. M., Higuchi, K., Hospattankar, A., Lackner, K., Leen, N., and Brewer, H. B., Jr. (1986) Proc. Natl. Acad. SC;. U. S: A. 83,8142-8146

20. Cladaras, C., Hadzopoulou-Cladaras, M., Nolte, R. T., Atkinson, D., and Zannis, V. I. (1986) EMBO J. 5,3495-3507

21. Yang, C.-Y., Chen, S.-H., Gianturco, S. H., Bradley, W. A., Snarrow. J. T.. Tanimura. M.. Li. W.-H.. Snarrow. D. A.. DeLoof, H., Rosseneu, M., Lee, F.-S., Gu, Z.-W:, Gotto, A. M.; Jr., and Chan, L. (1986) Nature 323, 738-742

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

Driscoll, D. M., Wynne, J. K., Wallis, S. C., and Scott, J. (1989) Cell 58,519-525

Gorski, K., Carneiro, M., and Schiber, U. (1986) Cell 47, 767- 776

Shapiro, D. J., Sharp, P. A., Wahli, W. W., and Keller, M. J. (1988) DNA 7, 47-55

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R. G., Horn, T. T., Mullis, K. B., and Erlich, H. (1988) Science 239,487-491

Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. N&l. Acad. Sci. U. S. A. 74,5463-5467

Davidson, N. O., Powell, L. M., Wallis, S. C., and Scott, J. (1988) J. Biol. Chem. 263,13482-13485

Tennyson, G. E., Sehatos, C. A., Higuchi, K., Meglin, N., and Brewer, H. B., Jr. (1989) Proc. N&l. Acad. Sci. U. S. A. 86, 500-504

Gualberto, J. M., Lamattina, L., Bonnard, G., Weil, J.-H., and Grienenberger, J.-M. (1989) Nature 341, 660-662

Covello, P. S., and Gray, M. W. (1989) Nature 341,662-666

by guest on June 11, 2018http://w

ww

.jbc.org/D

ownloaded from

S H Chen, X X Li, W S Liao, J H Wu and L Chanvitro coupled transcription editing.

RNA editing of apolipoprotein B mRNA. Sequence specificity determined by in

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